Actin is a contractile protein found in most cell types. Actin proteins are represented by similar but nonidentical isoforms which in warm blooded vertebrates are encoded by multigene families. Actin is expressed in a developmentally timed dependent and tissue specific manner, in which adult muscle tissues express exclusively the skeletal alpha actin gene as the predominant actin isoform. The nucleic acid sequence of skeletal alpha actin gene has been characterized in chicken, rat, mouse and human. Fornwald et al., Nucleic Acids Res., Vol. 10, pp. 3861-3876 (1982); French et al., Gene, Vol. 88, pp. 173-180 (1990); Zak et al., Nature, Vol. 298, pp. 857-859 (1982); Hu et al., Mol. Cell. Biol., Vol. 6, pp. 15-25 (1986) and Minty and Kedes, Mol. Cell. Biol., Vol. 6, pp. 2125-2136 (1986).
Once a gene has been characterized and sequenced it can be mapped to determine its protein coding and noncoding portions. RNA transcripts are initiated from a promoter region start site at the messenger RNA cap and continue through the protein coding region to the noncoding region involved with transcriptional termination. Post transcriptional processing of the RNA transcripts removes the noncoding introns to form a continuous coding sequence. Further, there is progressive trimming of the RNA transcript to the polyadenylation signals, found in the noncoding 3' untranslated portion of the processed messenger RNA. The levels of messenger RNA biosynthesis and accumulation is determined by parts of the unexpressed portion of the gene. For example the promoter, which is part of the unexpressed portion of the gene, is involved in determining gene expression. It regulates when and how the expressed gene is transcribed. The accumulation of RNA transcripts, however, may relate to the intrinsic stability of that RNA in a particular cell type. This intrinsic stability is dependent on sequences within the MRNA that provide stabilization. Thus, the ability to express a particular gene product is a balance between transcription rate and the stability of the MRNA transcript.
Expression of the actin gene family is tissue specific. The skeletal alpha actin protein is expressed primarily in cardiac and skeletal muscle tissues. Transient transfection experiments indicated that regions within the 200 bp promoter region are sufficient for tissue restricted expression in primary myoblasts. Bergsma, et al, Mol. Cell. Biol., Vol.6 pp. 2462-2475 (1986) and Chow and Schwartz Mol. Cell Biol., Vol.10 pp. 528-538 (1990). Further, the promoter region harbors conserved cis-acting elements to accurately initiate skeletal alpha actin transcripts from the bacterial reporter gene chloramphenicol acetyltransferase (CAT) in differentiating myoblasts. Grichnik et al. Nucleic Acids Res., Vol. 14, pp. 1683-1701 (1986). Transgenic mice with an integrated skeletal alpha actin promoter showed preferential expression of the CAT gene in myogenic tissues Petropulous et al. Mol. Cell. Biol., Vol. 9 pp. 3785-3792 (1989). It is also known that CAT activity is detectable as early as 10 days in the mouse embryo when the embryonic heart is first being formed. Further, CAT activity can be induced in newborn skeletal muscle. Thus, the skeletal actin promoter can switch transcriptional activity in mammalian systems, to provide muscle restricted expression. However, when measurements were made to compare the levels of CAT messenger RNA to that of the endogenous skeletal alpha actin messenger RNA (which in the adult muscle was found at levels over thousand fold higher than the embryo), the CAT messenger RNA could not be detected by standard RNA blotting techniques. These experimental results showed that even though small amounts CAT protein accumulated in skeletal muscle, the CAT messenger RNA never accumulated to the levels of RNA transcripts from the endogenous skeletal actin gene.
Current trends for optimizing gene transcription vectors have been directed towards increasing the transcriptional activity of the vector system. For example, Observations in the human Beta globin gene system indicated that expression in transgenic mice was never as high as the expression of the endogenous mouse Beta globin gene. Grosveld and co-workers Cell, Vol. 51, pp 975-985 (1987) It was determined that the sites surrounding the globin locus contain a number of DNAse hypersensitive sites that are termed the dominant control region. This region appears to act as tissue specific enhancers. When some of these sites were cloned into a minilocus gene it provided erythroid tissue specific expression in transgenic animals. Regulatory sequences similar to the dominant control regions have been described for human CD2. Greaves et al. Cell, Vol. 56, pp. 979-986 (1988). It is known that the avian skeletal actin promoter is as active as the SV40 promoter which is a standard for high levels of expression. Bergsma, et al, (1986). Hence, the skeletal actin promoter is not the cause for low levels of messenger RNA accumulation. Thus, the actin promoter is by itself insufficient to drive the expression of other gene products to accumulate at levels which are comparable to the intact skeletal alpha actin MRNA.
The rate of metabolic breakdown of MRNA molecules is an important factor in the regulation of gene expression. The rates of decay of individual MRNA species can affect strongly the steady state levels of these species in the cytoplasm. Consequently, the extent of expression of a given gene, as measured by the rate of synthesis of the corresponding protein, will be dependent to a large extent on the degree of stability of the MRNA derived from this gene. Messenger RNA from skeletal muscle was previously shown to be distributed into two populations with regard to its stability. Medford et al. J. Biol. Chem., Vol. 258, pp. 11063-11073 (1983). One MRNA population had a half life of less than 4 hours and the other population had a half-life of 17-to over 54 hours.
Comparison of the untranslated regions in vertebrate skeletal alpha, cardiac alpha, and beta actin MRNA has revealed regions of high sequence homology within the 3' untranslated portion of each of these actin isoformic MRNA and that this homology is greater among the alpha-cardiac and skeletal actin isoforms than between alpha striated actin and beta actin isoform MRNA. Mayer et al., Nucl. Acids Res., Vol 12. pp. 1087-1100 (1984); Ponte et al., Nucl. Acids Res., Vol. 12 pp. 1687-1696 (1984); Chang et al., Nucl. Acids Res., Vol. 13 pp. 1223-1237 (1985). In comparison, other vertebrate genes, such as those encoding insulin and prolactin share common coding regions but usually contain divergent 3' untranslated regions. The preservation of the 3' untranslated regions of the skeletal alpha actin gene in animal species ranging from birds to humans suggests that they have important biological roles. In the present invention, the incorporation of the myogenic specifies 3' untranslated region into recombinant DNA vectors for expressing polypeptides in muscle tissue was found to be highly advantageous, since it enhanced the polypeptide MRNA content in muscle by increasing stability.